Heart transplantation is often the only available therapeutic option in end-stage heart disease. To monitor graft survival, recipients endure frequent invasive endomyocardial biopsies which carry a significant risk of complication and are prone to sampling-errors. There is thus a compelling need to develop noninvasive, more predictive and quantitative diagnostic tools to monitor individual patients and to compare new treatment regimen in different patient cohorts. An increasing body of work suggests that macrophages are highly abundant in rejected allografts, and exhibit cardinal cellular and molecular functions. The overall goal of this proposal is to evaluate macrophages and macrophage-associated functions as in vivo imaging biomarkers of transplant rejection. Specifically, we will validate a) nanoparticles as reporters of phagocytic activity, b) optical prodrugs as sensors of cysteine protease activity, and c) myeloperoxidase-targeted magnetic resonance imaging agents. We will investigate which of the above imaging marker correlates most closely with the ex vivo gold standard (ISHLT-graded histology) and CD68 expression and if imaging can detect earliest forms of rejection and/or tolerance at sufficiently high sensitivity and specificity. We will test these MU-targeted cardiac imaging tools to non-invasively investigate 3 specific therapeutic approaches to treat transplant rejection. First we will benchmark macrophage imaging during the use of routine immunosuppressants (calcineurin inhibitor, antimetabolite agent and corticosteroids). Next we will investigate emerging tolerance regimen (induction of mixed chimerism by bone marrow co- transplantation). Finally, we will use mice deficient for macrophage recruiting cytokines and adoptive cell transfer to reprogram prevalent macrophage populations in the heart from the inflammatory M1 to reparative M2 subsets. These longitudinal studies will be used to validate functional macrophage-targeted agents for noninvasive heart transplant imaging.